The present invention relates to plant molecular biology. In particular, it provides compositions and methods useful for modulating fatty acid synthesis in plants.
Fatty acids (FAs) are the major constituents of acyl lipids in plant tissues. Acyl lipids are mainly present as triacylglycerols in the oil bodies of tissues which serve as food storage, such as seeds and the fleshy parts of fruits. These tissues are important commercial sources of fats and oils. Fatty acids are also found as glycolipids and phospholipids in other tissues, such as leaves, roots, or shoots, where they are integral components of the various cell membranes.
The principal FAs are saturated or unsaturated monocarboxylic acids with an unbranched even-numbered carbon chain. The main saturated FAs are lauric (12:0, i.e., C12 chain with no double bonds), myristic (14:0), palmitic (16:0), and stearic (18:0). The main unsaturated FAs are oleic (18:1), linoleic (18:2)and linolenic (18:3). Seed storage lipids accumulate mostly 16- and 18-carbon FAs. Oilseeds of the Cruciferae and a few other plants also accumulate C20 and C22 FAs, collectively referred to as very long chain fatty acids (VLCFAs) because of their relatively longer chain length compared to the more common FAs found in plants (see, in general, Stumpf, in Biochemistry of Plants, Vol. 9, Stumpf ed., Academic Press, New York, 1987) and Browse and Somerville, Ann. Rev. Plant Physiol. Plant Mol. Biol. 4:467-506 (1991).
The presence of VLCFAs in vegetable oils markedly affects their use. For example, erucic acid (22:1) has detrimental nutritional effects and, thus, is undesirable in edible oils. Rapeseed oil is naturally high in erucic acid, but through a concerted breeding effort, canola lines that are almost devoid of erucic acid have been developed (Loof and Appleqvist, in Rapeseed, Appleqvist and Ohlson, eds. Elsevier Publishing, 1972). On the other hand, vegetable oils high in erucic acid have found many industrial uses, including use as diesel fuel and as a raw material for an array of products, including paints, corrosion inhibitors, cosmetics, plastics, pharmaceuticals, and lubricants (Murphy, Tibtech 10:84-87 (1992).
The biosynthesis of saturated FAs having a carbon chain up to C18 proceeds in the chloroplast via the sequential condensation of C2 units from acyl thioesters. FA synthesis is initiated by the condensation of acetyl CoA and malonyl ACP, catalyzed by the recently discovered enzyme .beta.-ketoacyl synthase III (KASIII) (Jaworski et al., Plant Physiol. 90:41-44 (1989)). The enzyme ketoacyl synthase I is required for the elongation of saturated acyl-ACP from C4 to C16. The last elongation step in the chloroplast, from C16 to C18, is catalyzed by ketoacyl synthase II. Each condensation is followed by three enzymatic steps, which involve reduction and dehydration of the .beta.-ketoacyl derivative formed by the synthase and reduction of the double bond in the corresponding enoyl-ACP intermediate (Stumpf, 1987, supra).
Elongation of the FA carbon chain from C18 to C22 occurs outside the chloroplast by the sequential addition of two C2 moieties from malonyl CoA to a C18 carbon skeleton, a reaction catalyzed by a particulate acyl CoA elongase complex (Stumpf and Pollard, in High and Low Erucic Acid Rapeseed Oils, Kramer et al. eds. Academic Press, 1983). Whether the two elongation reactions are carried out by one or two different enzyme complexes is not clear (Taylor et al., Plant Physiol. 99:1609-1618 (1992)). The same four reactions described above for the biosynthesis of C18 FAs are involved in the further elongation of C18 in plants: (i) condensation of 18:1 CoA with malonyl CoA to form a .beta.-ketoacyl derivative, (ii) reduction and (iii) dehydration of the .beta.-ketoacyl derivative, and (iv) reduction of the double bond (Creach and Lessire JAOCS 70:1129-133(1993)). However, because of the difficulties in solubilizing membrane-bound enzymes, the elongase complex has not been well characterized. Elongases have been partially purified from several plants, including Allium porrum or leek (Bessoule et al., Arch. Biochem. Biophys. 268:475-484 (1989)), Lunaria annua or honesty (Fehling et al., Biochim. Biophys. Acta 1126:88-94 (1992)), and Brassica napus or rapeseed (Creach and Lessire, 1993, supra).
In Arabidopsis, mutations in a gene associated with fatty acid elongation, the FAE1 gene, result in a deficiency in acyl chain elongation activities from C18 to C20 and C20 to C22, and in highly reduced levels of seed VLCFAs (James and Dooner, Theor. Appl. Genet. 80:241-245 (1990); Lemieux et al., Theor. Appl. Genet. 80:234-240 (1990); James and Dooner Theor. Appl. Genet. 82:409-412 (1991); and Kunst et al. Plant Physiol. Biochem. 30:425-4343 (1992)).
FA biosynthetic genes have been isolated by the conventional biochemical approaches of purifying the corresponding enzyme in order to generate antibodies or oligonucleotides with which to probe cDNA libraries. Among them are genes for ACP (Schmid and Ohlrogge, Plant Mol. Biol. 15:765-778 (1990)), KASII (International Publications WO92/03564 and WO93/10240), KASIII (Tai and Jaworski, Plant Physiol. 103L1361-1367 (1993)), stearoyl-ACP desaturase (International Publication No. WO91/18985), acyl-ACP thioesterases (U.S. Pat. No. 5,298,421) enoyl-ACP reductase (Kater et al., Plant Molec. Biol. 17:895-909 (1991)), 3-ketoacyl-ACP reductase (Klein et al., Mol. Gen. Genet. 233:122-128 (1992)), acyl-ACP:glycerol-3-P acyl transferase (Weber et al., Plant Molec. Biol. 17:1067-1076 (1991)). Others have been isolated on the basis of DNA homology to previously cloned genes from related species, e.g., genes for stearoyl-ACP desaturases (Knutzon et al., Proc. Natl. Acad. Sci. USA 89:2624-2628 (1992)) or acyl-ACP:glycerol-3-P acyl transferases (Nishida et al., Plant Molec. Biol. 21:267-277 (1993)).
Genes encoding FA biosynthetic enzymes in Arabidopsis have also been isolated. Examples include the FAD3 gene encoding an endoplasmic reticulum (ER) 18:2 desaturase (Arondel et al., Science 258:1353-1354 (1992)), FAD3 (Yadav et al., Plant Physiol. 103:467-476 (1993)), and the FAD2 gene, which encodes another ER enzyme, an 18:1 desaturase (Okuley et al. The Plant Cell, 6:147-158 (1994)).
There have been no reports of the isolation of FAE1 genes. Isolation of these genes would be particularly useful in modulating fatty acid synthesis in plants. These and other advantages are provided by the present invention.